Cell Technology for Cell Products

Abstract

Bacteria and mammalian cells have been traditionally used as hosts for commercial recombinant protein production. In recent years, the insect cell-baculovirus system has emerged as a potentially attractive recombinant protein expression vehicle. This route is attractive because baculovirus-infected insect cells are able to perform post-translational modification while accommodating very abundant expression of recombinant protein. Although flow cytometry has been used widely for analysis of mammalian and microbial cell physiology and morphology, there is very little information on applications of this powerful and highly efficient technique in insect cell culture.\rHere we have compared cell ratiometric counts and viability of Sf-21 cell cultures using a flow cytometer to those determined by more traditional methods using a haemocytometer and the trypan-blue exclusion dye. There was good agreement between the two counting methods but the former technique proved to be a more reliable and statistically robust viability indicator (stains used: propidium iodide and calcein AM).\rFlow cytometry has also been used to monitor various parameters during fermentations of Sf21 cultures infected with the recombinant Autographa californica Nuclear Polyhedrosis Virus (AcNPV). This recombinant baculovirus contains the inserted nucleic acid sequence amFP486 coding for AM-Cyan coral protein, which emits natural green fluorescence. A good correlation has been obtained between parameters such as mean green fluorescence and AmCyan positively stained cells linked to cell viability. Additionally, DNA content, cell size and granularity have proven to be good indicators of baculovirus infection.\rWe have also simplified and optimised methods of cell treatment prior to flow cytometric analysis.